Affiliation: Nuffield Department of Medicine, University of Oxford, The Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, UK. lian.lee@ndm.ox.ac.uk

ABSTRACT

Background: Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.

Method: Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.

Results: CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.

Conclusions: Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.

Figure 2: Control of mycobacterial growth after aerosol M. tuberculosis challenge of Ad85A immunized mice. BALB/c mice were immunized with Ad85A i.d. or i.n. and challenged 4 weeks later with M. tuberculosis by aerosol. Mice were sacrificed 6 weeks later and mycobacterial burden in the lungs (A) and spleen (B) determined. The results show the log CFU in each mouse and the mean for each group and are representative of at least 2 independent experiments. The data were analyzed using the Kruskal-Wallis test (p = 0.007 comparing all groups), followed by Dunn's multiple comparison test, which returned p-values of < 0.05 for comparisons between naïve vs. i.n. and i.d. vs. i.n. groups. * indicates p < 0.05 compared to naïve or i.d.

Mentions:
In agreement with previous reports [2,5,6], mice immunized with Ad85A i.n. developed a strong 85A-specific T cell response in the lung and a much weaker splenic response, while Ad85A i.d. immunized mice make a stronger spleen and weaker lung response (Figure 1). Ad85A i.n. mice reduced the lung mycobacterial load by ~ 1 log compared to naïve mice (Figure 2A), a protective effect comparable to BCG [5,12], while Ad85A i.d. immunization did not reduce the bacterial load either in the lungs or spleen (Figure 2B). The responses induced by Ad85A in BALB/c mice are dominated by CD8 T cells (Figure 1). In C57BL/6 mice Ad85A immunization induces a CD4 response and no consistent statistically significant protection against pulmonary M. tuberculosis challenge is obtained. However in BALB/c mice it has been demonstrated that antigen-specific CD8 T cells in the lung are critical for protection against pulmonary infection with M. tuberculosis in this immunization model [7,13]. Therefore we wished to determine if CD8 T cells from the lungs and spleens of BALB/c mice differed. Mice were immunized i.n. or i.d. with Ad85A and CD8 T cells isolated from the lungs (lung i.n. or i.d.) or spleens (spleen i.n. or i.d.) 3 weeks post-immunization, close to the peak of the cytokine response [5]. Purification by positive selection resulted in CD8 T cell populations which were 80-86% pure from lungs and 86-90% pure from spleens. Total RNA was prepared from cells isolated in 3 independent experiments, amplified and global genome analysis was performed using Illumina microarrays. The expression profiles of spleen and lung CD8 T cells were compared. Transcripts with ≥ 2 fold difference in signal intensity with a p-value of ≤ 0.05 were considered for further analysis, apart from the comparison between spleen i.n. and spleen i.d. where transcripts with ≥ 1.5 fold difference were considered.

Figure 2: Control of mycobacterial growth after aerosol M. tuberculosis challenge of Ad85A immunized mice. BALB/c mice were immunized with Ad85A i.d. or i.n. and challenged 4 weeks later with M. tuberculosis by aerosol. Mice were sacrificed 6 weeks later and mycobacterial burden in the lungs (A) and spleen (B) determined. The results show the log CFU in each mouse and the mean for each group and are representative of at least 2 independent experiments. The data were analyzed using the Kruskal-Wallis test (p = 0.007 comparing all groups), followed by Dunn's multiple comparison test, which returned p-values of < 0.05 for comparisons between naïve vs. i.n. and i.d. vs. i.n. groups. * indicates p < 0.05 compared to naïve or i.d.

Mentions:
In agreement with previous reports [2,5,6], mice immunized with Ad85A i.n. developed a strong 85A-specific T cell response in the lung and a much weaker splenic response, while Ad85A i.d. immunized mice make a stronger spleen and weaker lung response (Figure 1). Ad85A i.n. mice reduced the lung mycobacterial load by ~ 1 log compared to naïve mice (Figure 2A), a protective effect comparable to BCG [5,12], while Ad85A i.d. immunization did not reduce the bacterial load either in the lungs or spleen (Figure 2B). The responses induced by Ad85A in BALB/c mice are dominated by CD8 T cells (Figure 1). In C57BL/6 mice Ad85A immunization induces a CD4 response and no consistent statistically significant protection against pulmonary M. tuberculosis challenge is obtained. However in BALB/c mice it has been demonstrated that antigen-specific CD8 T cells in the lung are critical for protection against pulmonary infection with M. tuberculosis in this immunization model [7,13]. Therefore we wished to determine if CD8 T cells from the lungs and spleens of BALB/c mice differed. Mice were immunized i.n. or i.d. with Ad85A and CD8 T cells isolated from the lungs (lung i.n. or i.d.) or spleens (spleen i.n. or i.d.) 3 weeks post-immunization, close to the peak of the cytokine response [5]. Purification by positive selection resulted in CD8 T cell populations which were 80-86% pure from lungs and 86-90% pure from spleens. Total RNA was prepared from cells isolated in 3 independent experiments, amplified and global genome analysis was performed using Illumina microarrays. The expression profiles of spleen and lung CD8 T cells were compared. Transcripts with ≥ 2 fold difference in signal intensity with a p-value of ≤ 0.05 were considered for further analysis, apart from the comparison between spleen i.n. and spleen i.d. where transcripts with ≥ 1.5 fold difference were considered.

Affiliation:
Nuffield Department of Medicine, University of Oxford, The Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, UK. lian.lee@ndm.ox.ac.uk

ABSTRACT

Background: Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.

Method: Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.

Results: CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.

Conclusions: Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.